10 February 2010. The hypothesis that schizophrenia results from underachieving N-methyl D-aspartate (NMDA) receptors or, at the very least, disturbed glutamate neurotransmission, has spurred researchers to seek ways to tweak NMDA-R function. Two new studies in Nature Neuroscience may clarify some of the mechanisms involved. Akira Sawa and colleagues find that DISC1, a gene associated with schizophrenia and other major mental disorders in a large Scottish family, helps regulate the plasticity of dendritic spines in response to NMDA-receptor activation. Stimulating NMDA receptors causes DISC1 to limit kalirin-7’s access to the GTPase Rac1, thereby affecting the formation of dendritic spines. In other work, Paul Roche, Katherine Roche, and others report that SNAP23, a relative of the schizophrenia-associated synaptic trafficking protein SNAP25, regulates NMDA receptors in the post-synaptic membrane. Together, the studies offer new information on the details of NMDA receptor regulation and potential insights into how that function might be altered, or treated, in schizophrenia.

In the brain, most excitatory synaptic neurotransmission takes place on dendritic spines, but it has been reported that patients with schizophrenia have too few of these structures (see SRF related news story), perhaps due to DISC1 abnormalities (see SRF related news story). Sawa, first author Akiko Hayashi-Takagi, both of Johns Hopkins University in Baltimore, Maryland, and their colleagues sought to understand how DISC1 governs the building of these spines. Their paper appeared on February 7 in an advance online publication of Nature Neuroscience.

To study the effects of DISC1, the researchers injected lentivirus carrying two short-hairpin RNAs (shRNAs) into the prefrontal cortex of adult rats to knock down expression of DISC1 and its main isoforms. Two days later, dendritic spines had multiplied and grown. Furthermore, expression of the AMPA-type glutamate receptor subunit GluR1 on the cell surface had increased, along with the frequency of miniature excitatory post-synaptic currents (mEPSCs).

Hayashi-Takagi and colleagues used immunoprecipitation to identify DISC1’s binding partners in cortical neurons and rat brains. As they predicted, DISC1 interacted with kalirin-7 and PSD-95. It did not interact with guanine nucleotide exchange factors other than kalirin-7.

The researchers did not stop there. To learn which domain of DISC1 binds with kalirin-7, they studied the effects of deleting various DISC1 segments. They noted that binding did not occur if DISC1 was missing a certain sequence of 44 amino acids. Overexpressing DISC1 reduced spine size in neurons, but only if it contained that key segment. These results support the idea that DISC1 and kalirin-7 binding regulates spines.

Since NMDA receptor activation launches kalirin-7 signaling, the researchers applied electroconvulsive treatment to activate neurons in homogenized brain tissue. This lessened interactions of kalirin-7 with PSD-95, kalirin-7 with DISC1, and DISC1 with PSD-95. To confirm that the decreased binding resulted from specific activation of NMDA receptors, the investigators also targeted the receptors by withdrawing the inhibitory influence of amino-5-phosphonovaleric acid. This tactic destroyed the complex that DISC1 had formed with PSD-95 and kalirin-7. With kalirin-7 now freed from DISC1’s grip, it triggered Rac1 to control spine formation. In the longer term, the investigators showed that disruption of DISC1 expression led to spine shrinkage in rat primary cortical neurons, which could be rescued by full-length DISC1 but not the kalirin binding domain mutant.

Controlling traffic
Changes in SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins, which regulate vesicle trafficking and neurotransmitter release, might also help explain the synaptic abnormalities seen in schizophrenia (for a review, see Johnson et al., 2008). Studies show altered levels of synaptosomal-associated protein 25 (SNAP25), in various brain regions in the disorder (see Corradini et al., 2009; also see SRF related comments). Mice induced to express mutant human DISC1 show decreased SNAP25 levels and a schizophrenic phenotype (Pletnikov et al., 2008). Furthermore, polymorphisms in the gene encoding SNAP25 have been associated with schizophrenia in some studies (see SZGene entry for SNAP25).
Prior research finds SNAP25 (see SRF related news story) expression mainly in presynaptic membranes in brain neurons. There it forms part of the assembly that moves vesicles holding neurotransmitter to the cell membrane. By binding with other SNARE proteins, it joins the vesicle membrane to the cell membrane, releasing neurotransmitter into the synapse.

Unlike SNAP25, the structurally similar SNAP23 appears throughout the body (Ravichandran et al., 1996), including the brain. Paul Roche, Katherine Roche, and their colleagues at the National Institutes of Health in Bethesda, Maryland, raise an interesting question: If SNAP25 abounds in the brain and binds relatively well with other SNARE proteins, what is the similar SNAP23 doing there? Their paper in Nature Neuroscience, published online on January 31, finds SNAP25 only at presynaptic sites in the neuron, while its cousin works the other side of the synapse. The researchers, including first author Young Ho Suh, show that SNAP23 governs the transport and function of NMDA receptors.

Different neighborhoods
Suh and colleagues started by seeing where the two proteins appeared in rat hippocampal neurons in culture. After labeling the cells with antibodies for SNAP23 and SNAP25, they found SNAP25 in the axons and SNAP23 in the cell body and dendrites. Indeed, the two proteins seemed to have staked out their own separate turfs. As Suh and colleagues write, “The exclusive localization of SNAP23 on dendrites suggests that SNAP23 is involved in post-synaptic membrane trafficking events.”

To follow up on this finding, Suh and colleagues differentially centrifuged brain homogenate from rats to see which cell parts associated with the two proteins. SNAP25 fell out with the fraction that was enriched with synaptic vesicles. In contrast, SNAP23’s distribution echoed that of elements of the post-synaptic density. It showed up at the same sites as post-synaptic density-95, as well as NMDA receptor subunits NR2A and NR2B.

The researchers used light and electron microscopy to learn more about SNAP23’s presence within neurons. It revealed SNAP23 enrichment near NMDA and AMPA glutamate receptors, but not at inhibitory synapses.

Different jobs
Next, the researchers produced genetically altered mice that had half the normal levels of SNAP23. On the surface of their cortical neurons, the altered mice made less of the NMDA receptor subunits NR1 and NR2B than wild-type mice did. In contrast, their surface expression of AMPA receptor subunits, a GABA receptor, and a presynaptic metabotropic glutamate receptor seemed normal. Suh and colleagues write, “These data indicate that reduced expression of endogenous SNAP23 regulates the surface expression of NMDA receptors in neurons.”

Yet, the possibility remained that SNAP25 might do the same thing. To compare the two proteins’ effects, the researchers used lentivirus to deliver shRNA that would curb production of SNAP23 or SNAP25. Targeting SNAP23 in this way curbed expression of NMDA receptor subunits NR2A, NR2B, and NR1 at the neuronal surface. It also modestly reduced surface expression of two AMPA receptor subunits. In contrast, the SNAP25 knockdown did not affect NMDA or AMPA receptor subunits.

A final test of the proteins’ roles involved taking voltage-clamp recordings of whole pyramidal neurons from the hippocampal CA1 region. Cells that expressed SNAP23, but not SNAP25 shRNA showed weakened NMDA-evoked currents. Not only did SNAP23 regulate these currents, it also affected NMDA excitatory post-synaptic currents, apparently by controlling the number of NMDA receptors at synapses.

The study by Suh and colleagues suggests that SNAP23 controls the trafficking and behavior of post-synaptic NMDA receptors. Whether the protein causes their dysfunction in schizophrenia (see SRF Current Hypotheses by Bita Moghaddam and by Daniel Javitt) remains to be seen. While some studies have looked at SNAP25 in regard to schizophrenia and related phenotypes, a PubMed search using the terms “SNAP23” and “schizophrenia” came up empty. That may change. Researchers have been seeking promising targets to treat NMDA dysfunction in the disease (see SRF related news story; SRF related news story). Knowing more about the mechanisms involved may help.—Victoria L. Wilcox.

The newly published paper by Katherine Roche and Paul Roche reports SNAP-23 expression in neuron dendrites and examines the possible role of this neuronal SNAP-23 protein. To this point, SNAP-23 has traditionally been discussed in reference to vesicle trafficking in epithelial cells (see Rodriguez-Boulan et al., 2005 for review), so it is of interest to determine the function of SNAP-23 in neurons. Suh et al. report that surface NMDA receptor expression and NMDA-mediated currents are inhibited following SNAP-23 knockdown. Further, SNAP-23 knockdown results in a specific decrease in NR2B subunit insertion; previously, the NR2B subunit has been reported to preferentially localize to recycling endosomes compared to NR2A (Lavezzari et al., 2004). Given these findings, it is reasonable to conclude that SNAP-23 may be involved in maintaining NMDA receptor surface expression possibly by binding to NMDA-specific recycling endosomes.

Interestingly, there is recent evidence that PKC-induced NMDA receptor insertion is mediated by another neuronal SNARE protein; postsynaptic SNAP-25 (Lau et al., 2010). It is possible that activity-induced NMDA receptor trafficking is mediated by SNAP-25, while baseline maintenance of NMDA receptor levels relies on SNAP-23. Other evidence to suggest a strictly regulatory role for SNAP-23 in neuronal NMDA insertion is the finding that activity-dependent receptor insertion from early endosomes has previously been reported to be restricted to AMPA-type glutamate receptors (Park et al., 2004). However, it is possible that activity-induced insertion of AMPA receptors occurs via a distinct endosome pool than NMDA receptors; AMPA and NMDA receptor trafficking has been reported to proceed by distinct vesicle trafficking pathways (Jeyifous et al., 2009).

Although SNAP-23 may not be involved in activity-dependent early endosome receptor trafficking, it is possible that SNAP-23 operates in other pathways linked to activity-induced NMDA receptor trafficking. For instance, SNAP-23 may be the SNARE protein by which lipid raft shuttling of NMDA receptors occurs. SNAP-23 has been found to preferentially associate with lipid rafts over SNAP-25 in PC12 cells (Salaün et al., 2005). As well, NMDA receptors have been found to associate with lipid raft associated proteins flotilin-1 and -2 in neurons (Swanwick et al., 2009). Lipid raft trafficking of NMDA receptors to post-synaptic densities has been reported to follow global ischemia (Besshoh et al., 2005), and the possibility remains that under certain circumstances, NMDA trafficking occurs by lipid raft association to SNAP-23.

Taken together, the discovery of post-synaptic SNARE proteins offers several avenues of research to determine their roles and functions in glutamatergic synapse organization. Further, investigating disruption of synaptic receptor organization presents several possibilities for potential etiologies of disorders linked to compromised glutamate signaling like schizophrenia.